This invention provides a novel method for purifying Invaplex, a novel composition comprising at least one invasin protein i.e. a protein essential for the process by which a bacterium enters a host cell, and lipopolysaccharide from invasive gram negative bacteria. The composition of the present invention can be used as an adjuvant for vaccines, biochemical, or other substances, and as a diagnostic tool.
Bacillary dysentery is caused by members of the genus Shigella and also enteroinvasive Escherichia coli (EIEC). Shigellosis is found in all parts of the world with developing countries accounting for the large majority of cases. A recent report in the Bulletin of the World Health Organization estimates that in developing countries, for children 0-4 years old, there were 113 million Shigella episodes per year and an additional 50 million cases per year in all other age groups. In industrialized countries it is estimated that there are approximately 1.5 million cases of shigellosis per year (Kotloff et al. 1999, WHO 77, 651-666). The rampant occurance of antibiotic resistance in Shigella spp. and the high incidence of this disease underscores the need for a vaccine against this human pathogen. However at the present time a vaccine is not commercially available for bacillary dysentery.
The pathogenesis of Shigella is attributed to this organism""s ability to invade, reside, and replicate intracellularly within the colonic epithelium. The invasion of host cells by Shigella spp. is a complex multifactorial event in which many different bacterial proteins are involved. Many of the genes for key Shigella virulence proteins are encoded on a large 140 Mdal plasmid. Several of the plasmid encoded proteins called the invasion plasmid antigens (IpaA, IpaB, IpaC, and IpaD proteins) (Buysse et al., 1987, J. Bacteriol. 169, 2561-2569) are essential virulence factors. Similar proteins, called Sip proteins, are made by members of the genus Salmonella (Kaniga et al., 1995, J. Bacteriol. 95, 3965-3971). Upon contact or attachment to host cells, the Shigella invasins induce a phagocytic event which results in engulfment and internalization of the bacterium by the host cell. Recent reports have identified that IpaB and IpaC form a complex that can be found in the growth medium of Shigella cultures (Menard et al., 1998, EMBO J 13, 5293-5302; Watari et al. 1995, EMBO J 14, 2461-2470). The components of this complex are involved in the invasion process, but the actual mechanisms have not been defined (Menard et al., 1994, Cell 79:515-525). In addition, purified IpaC has been shown to bind to host cells and participate in the uptake of avirulent shigellae by host cells (Marquart et al., Infect Immun. 64:4182-4187, 1996). Involvement in the attachment process and a possible role in the induction of phagocytosis suggests that the invasins or the invasin complex (if it can be isolated) would have adjuvant properties similar to cholera toxin, which also exhibits binding and internalization properties. Furthermore, IpaB, IpaC and IpaD, along with LPS are known major antigens that individuals respond to after infection with shigellae (Li et al. 1993, Scand. J. Infect. Dis. 25, 569-577; Oaks et al., 1986, Infect. Immun. 53, 57-63; van DeVerg et al. 1992, J. Infect. Dis. 166, 158-161). Monkeys or humans infected with shigellae produce antibodies predominantly to IpaB and IpaC, and also produce antibodies at high frequencey to IpaA, IpaD and VirG (another plasmid encoded virulence protein involved in intercellular spreading) (Oaks et al., 1986, supra; Lett et al., 1989, J. Bacteriol. 171, 353-359). It is not known if the immune response to the Shigella invasins or more specifically to the invasin complex is crucial to protective immunity.
Many proteins, carbohydrates, and even nucleic acids are not able to induce an immune response in animals unless they are coadministered with an adjuvant. An adjuvant, by definition, is an agent that increases specific immune responses to an antigen that would otherwise be incapable of eliciting an immune response. There are many different types of adjuvants, including aluminum salts (alum), cytokines, surface active agents, and various bacterial products such as Fruend""s adjuvant or Vibrio cholerae and E. coli enterotoxins. Effectiveness of an adjuvant is often dependent upon its ability to stabilize epitope conformation, preserve the antigen from rapid clearance and degradation, and to target the antigen to surface receptors on antigen-presenting cells. Of all the established adjuvants only cholera toxin (CT) and the E. coli labile toxin (LT) are purified proteins capable of inducing a potent humoral and mucosal immune response, specifically IgA. In the case of CT, a Th2 T cell response occurs which is characterized by increased levels of IL-4 and IL-5. Among other things, these cytokines lead to increased levels of certain classes of immunoglobulins. Th2 responses are often characterized by higher levels of IgA and the IgG1 subclass of IgG. The ability to stimulate a mucosal immune response is a highly desirable property, as most adjuvants are used primarily for systemic immunizations and produce little to no secretory immunity. Unfortunately, CT and LT are toxic molecules and have required genetic modifications to render these adjuvants safe and effective.
In contrast to adjuvants, immunological xe2x80x9ccarriersxe2x80x9d are usually proteins that have haptens or weakly immunogenic molecules covalently attached or genetically incorporated. In addition, living organisms are often used to carry or deliver foreign antigens to the host. Carriers provide T cell help to the antigen and thereby promote the immune response to the antigen.
A safe, effective mucosal adjuvant is desperately needed to promote the immune response to a multitude of new antigens becoming available due to recombinant DNA technology. Delivery of these antigens in a manner which promotes a protective immune response to pathogens depends in large parton an effective adjuvant.
One of the most elusive aspects to understanding how an infected host responds to a pathogen is the indentification of a specific immune response which correlates with protection against future disease. The quest for vaccines to a multitude of pathogens often goes astray because this one single, basic understanding of the immune response to a particular pathogen is not known. In the case of most enteric pathogens this is true.
The primary method for identifying correlates of a protective immune response is the development of a laboratory assay to assess the immune response in infected individuals. A multitude of different assay formats can be used but more often than not it is the antigen used in these assays which is critical. Assays for measuring antibodies, circulating antibody-secreting cells, T cells, cytokines, and other immune effectors are possible.
In the case of Shigella an assay which measures an antibody response which correlates with a protective immune response has not been developed. ELISAs are available for LPS and also the virulence proteins (Ipa proteins) of shigella. However, it has not been possible with any of these assays to demonstrate that a positive antibody response correlates with protection from future disease. The development of an assay which measures an antibody response that correlates with protection will expedite the development and testing of Shigella vaccines. It will also aid in the assessment of a person""s immune status with respect to Shigella.
Therefore, there is a need for a diagnostic tool which detects different gram negative bacteria and can measure an antibody response which correlates with protection against future disease.
The present invention fulfills the needs described above. In this application is described a novel composition comprising at least one invasin protein, a protein essential for bacterial invasion of a host cell, such as IpaA, IpaB, IpaC, and IpaD for Shigella. The invasin protein or proteins of the present invention are complexed with lipopolysaccharide (LPS). The complex of invasion protein or proteins and LPS is in a native conformation and has been termed Invaplex. The Invaplex described below is not only effective as a vaccine against gram-negative bacterial infection but it can also serve as a mucosal adjuvant and a diagnostic tool for detecting antibody response which correlates with protection against future infection.
Our initial experiments were aimed at isolating and purifying IpaC from a water extract of Shigella, a gram-negative bacteria. Usually, IpaC is extracted from growth culture medium. We chose to use the water extract, i.e. the solution resulting from incubating the bacteria with shaking in sterile water, because we hypothesized that the quantity of IpaC would be greater in such an extract. To our knowledge, no protein involved in the invasiveness of gram negative bacteria has been previously isolated from a water extract of gram negative bacteria. To our suprise, when water extract was subjected to various separation techniques such as gel filtration and ion-exchange chromatography, we found that whenever we could detect IpaC from the water extract, we also detected IpaB, IpaD and LPS in the same fractions. We proceeded to design a method to isolate this complex and characterize it. We have developed a method for purifying the Invaplex from intact invasive Shigellae or enteroinvasive E. coli (see FIG. 1 for general overview). Briefly, the Invaplex preparations are isolated from virulent, invasive shigellae. A crude mixture is extracted from the shigellae with water. The water extract consists of many proteins and lipopolysaccharide (LPS). The water extract material is then applied to a FPLC ion-exchange column which resolves two key protein peaks, called Invaplex (invasin complex) 24 and Invaplex 50. Fractions containing Invaplex 24 and Invaplex 50 are collected. We found that the complex was composed of many proteins, including IpaB, IpaC, IpaD in addition to LPS. The Invaplex 24 and Invaplex 50 preparations containing Ipa proteins and the LPS form a structure in a completely native configuration and environment. If such a structure is used to immunize animals, it will lead to an immune response directed against a native structure presented by gram-negative bacteria during infection. Mice and guinea pigs immunized with the Invaplex preparations showed a marked serum IgA and IgG response to several different antigens (including the water extract antigen, IpaC and LPS) present in the Invaplex 24 and Invaplex 50 preparations. The two Invaplex preparations were similar in that they both primed the mucosal immune system, but differed inthe specificity of the immune response generated. The animals were protected from challenge with gram negative bacteria and immunization with either Invaplex caused no visible distress to the animals. In addition, the highly immunogenic Invaplex can also serve as a very effective mucosal adjuvant very likely due to its ability to interact with the surfaces of host cells and present antigen to the immune system.
Our unpublished data where serum antibodies from recent vaccinees were found to be reactive with the Shigella water extract but poorly reactive with purified Ipa proteins indicates that the Invaplex may be recognized differently (conformationally) than the individual proteins. This suggested that a population of antibodies which specifically react with the complex and not the individual components are produced during an infection and may not be measured in an ELISA using individual purified proteins.
The Invaplex diagnostic assay of the present invention will measure a population of antibodies reactive with a virulence structure. Purified components of the invasin complex such as pure Ipa proteins or pure LPS, are unable to detect this population of antibodies.
Therefore, the present invention relates to a purified composition comprising Invaplex of gram-negative bacteria and methods of using the purified Invaplexes as adjuvants or diagnostic tools. The inventors have purified the Invaplex from two genera of gram-negative bacteria and determined that the complex comprises major antigenic proteins, the invasin proteins (e.g. for Shigella, IpaB, IpaC, and IpaD) as well as LPS. A novel method for purifying the Invaplex from intact bacteria is described.
The Invaplex containing invasin proteins and the LPS forms a structure in a completely native configuration and environment. This results in the ability to react with antibodies that are directed primarily toward epitopes involving the intact structure in addition to antibodies reactive with epitopes that are maintained on purified individual invasin proteins.
Experiments have shown that the Invaplex is also an effective adjuvant which results in very little reactogenicity or toxicity in addition to stimulating a potent mucosal and serum immune response when administered along with the desired antigen.
Therefore, it is one object of the present invention to provide a novel method for the isolation and purification of the Invaplex from gram negative bacteria.
It is another object of the present invention to provide a composition comprising isolated purified Invaplex as a diagnostic tool for detecting gram negative bacteria used as the source of the purified Invaplex.
It is yet another object of the present invention to provide a diagnostic assay for detecting gram-negative bacterial infection having the steps of contacting a sample from a subject suspected of having a gram negative bacterial infection with a purified Invaplex and detecting the presence or absence of a complex formed between the Invaplex and antibodies specific therefor, wherein the presence of a complex indicates presence of gram negative bacterial infection.
Further objects and advantages of the present invention will be clear from the description and claims that follow.